Project Summary Microfluidics, and miniaturization in general, is a powerful tool to enhance the performance of chemical analysis systems. Advantages include an increase in analysis speed, high-throughput parallel sample processing, and drastic reductions in sample, reagent, and power consumption. As such, microfluidic systems are the core technologies in many ?next-generation? separation, mass spectrometry, and DNA sequencing systems. Over the past decade, I have acquired theoretical and practical expertise in both microfluidic and chemical analysis technology development and came to The Scripps Research Institute (TSRI) to pursue application-driven research. To date, this research has largely focused on using the skills that I've already acquired to engineer a next-generation miniaturized droplet-based platform for compound screening. This integrated system distributes hundreds of thousands of DNA-encoded one-bead-one-compound (OBOC) combinatorial library beads into picoliter-scale droplets, precisely doses each droplet with UV light to liberate compound from the bead surface, performs assay incubation, and quantitates assay readout for hit identification and sorting. Screening an OBOC library with > 105 members requires only hours and < 200 L of assay reagents. Over the past two years, I have identified goals for my career, but I also realize that I do not currently possess the skills and scientific knowledge to accomplish them. My main goal is to transition the microfluidic droplet- based compound screening technology that I have helped develop into a start-up company. This company will focus on screening DNA-encoded compound libraries against viral and bacterial targets. The poor return on investment in such therapeutic areas has forced large pharmaceutical companies to divest from the space, but miniaturized high-throughput screening (HTS) technology can make such efforts cost-effective again and reinvigorate the drug pipeline for diseases that desperately need it. The K25 career development award is an excellent vehicle to supplement my expertise in microfluidics and analytical chemistry with training in molecular biology, organic chemistry, and assay development that is prerequisite for spearheading independent small molecule discovery efforts. To acquire these skills, I propose to generate a DNA-encoded OBOC combinatorial library that explores diversification of the ADEP 1 scaffold, an acyldepsipeptide natural product with antibiotic activity against Gram-positive bacteria, and screen it for both biochemical activity and whole-cell activity. Screening a large library of related compounds in orthogonal modes should provide insight into ?rules of penetration,? which can inform the design of novel synthetic scaffolds for anti-infective discovery. I have selected Brian M Paegel (TSRI) and Tom Kodadek (TSRI) as my mentor and co-mentor for this project. Prof. Paegel will provide me with extensive training in molecular biology and assay development techniques, and Prof. Kodadek is an invaluable resource who will guide design and synthesis of the OBOC library. TSRI provides an ideal environment for the project execution: TSRI houses a large HTS facility with experts in screening and assay development, TSRI's Infectious Diseases department has numerous world-class researchers, and proteomics, genomics, and cell-based screening core facilities are available for support. This proposal integrates key advances in DNA-encoded solid-phase synthesis (DESPS) and droplet microfluidics to form a distributable, highly efficient drug discovery platform. I will generate a combinatorial library of > 200,000 ADEP analogs that incorporates both natural and unnatural building blocks. Monomer coupling reactions will be evaluated for DNA compatibility, product yield, and purity. I will develop a biochemical activity assay that detects ADEP-mediated activation of its target ClpP using in vitro transcription and translation (IVTT) of both ClpP and the assay probe, GFP. Sufficient protein for > 106 droplet reactions can be generated quickly, inexpensively, and without cell culture. The ADEP analog OBOC library will be screened for activity against ClpP in picoliter-scale droplets using the developed assay. I will also screen the ADEP library for activity in a whole-cell bead diffusion assay. Separately, hit beads from each screen will be pooled, their DNA encoding tags amplified and their structures elucidated via next-generation sequencing. Hits will be resynthesized and validated in microplate-based biochemical activity assays and whole-cell viability assays. Lastly, hits will be ranked according to activity and comparative SAR between the hits for each screen should identify physicochemical parameters that aid in cell penetration.